Donor-specific tolerance would obviate the need for chronic immunosuppressive therapy in organ graft recipients, and might be necessary to overcome the formidable immunologic barriers to xenografts. We aim to induce T cell and B cell tolerance across a discordant species barrier (pig to mouse). We will first determine the ability of mixed bone marrow chimerism to induce tolerance among pre-existing and newly developing B cells producing NAb against the alpha1,3-galactosyl specificity that is recognized by most human anti-pig Nab. For this purpose, we will transfer alpha1,e-gal+ bone marrow,-alone or with alpha1,3-gal deficient mice receiving lethal and non-lethal conditioning regimens. In addition, alpha1,3-gal deficient, immunodeficient SCID-NOD mice will receive human B cells to determine which subsets produce anti- gal NAb, the effect of these NAb on porcine marrow engraftment and the ability to porcine chimerism to induce tolerance among anti-gal producing human B cells. We will also utilize the swine to SIC-NOD mouse BMT model to determine which cellular components of the human immune system (T cells subsets and NK cells) resist engraftment of swine BMC. Finally, we will induce pig-specific T and B cell tolerance in immunocompetent mice. Discordant pig-specific skin graft tolerance can be induce in normal mice by host thymectomy, mAbs to deplete recipient T cells, and replacement of the host thymus with a pig thymus, in which tolerant, immunocompetent mouse T cells develop. Pig cytokine transgenic mice will receive porcine thymus grafts with various sources of pig hematopoietic cells in an attempt to acheive hematopoietic chimerism. We will evaluate chimeric recipients for pig-specific natural antibody tolerance. We will ultimately attempt to acheive porcine chimerism in euthymic porcine cytokine transgenic recipients. Other studies will involve a species combination in which, like the pig-human combination, cell-cell interactions required for cellular immune responses are largely intact. In the concordant rat>mouse combination, marrow engraftment can be achieved following conditioning with a mild, non-myeloablative regimen. We will evaluate the role of gammadelta T cells and of alphabeta T cells that express NK cell markers in resisting xenogeneic marrow engraftment. By elucidating the components of the immune system that resist xenogeneic marrow engraftment, and in conjunction with results obtained in Project 3, these studies will help to overcome the xenogeneic transplantation barrier. Translation of results in this project to pre- clinical studies will advance the ability to induce donor-specific tolerance to pigs in humans.